FplA From Fusobacterium nucleatum is a Type Vd autotransporter phospholipase with a proposed role in altered host signaling and evasion of autophagy

Fusobacterium nucleatum is a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection, F. nucleatum lacks Type I, II, III, IV, and VI secretion. By contrast, F. nucleatum strains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of a F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined in F. nucleatum virulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with an fplA gene deletion strain, encompass a powerful set of tools for the future analysis of FplA in vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases. IMPORTANCE F. nucleatum is an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme from F. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and for F. nucleatum, in colorectal carcinogenesis.


INTRODUCTION
addition of whole live bacteria to a reaction containing the fluorogenic substrate  resulted in cleavage of the lipid substrate and a subsequent increase in fluorescence, 277 which was inhibited by the addition of MAFP (Fig. 4B). To further prove that full-length 278 FplA is expressed on the surface of E. coli, we confirm that treatment with the non-279 specific and cell-impermeable protease, Proteinase K (PK), cleaves FplA from the 280 surface, but does not cleave the cytoplasmic control GAPDH (Fig. 4C-D).  Table S2) that we developed from a Clostridium shuttle vector 53 using a 301 recombination method previously established for F. nucleatum 52 was used to create a 302 ΔfplA strain (Gene HMPREF0397_1968) (Strain DJSVT01, Table S1) marked with 303 chloramphenicol resistance (Fig. 5A-B). We verified by PCR that the fplA gene was 304 disrupted by the chromosomally-inserted plasmid, and further showed expression of the 305 protein had been abolished by a fluorescent probe and western blots probed with an 306 anti-FplA antibody. As phospholipases have been shown to play a role in bacterial 307 membrane maintenance, we tested F. nucleatum 23726 ΔfplA for changes in growth 308 rates and cell size, and found that when compared to wild-type F. nucleatum 23726, 309 there were no changes in these physical parameters when grown under standard 310 laboratory conditions (Fig. S5A-D). domains) around 25-30 kDa for strains 10953, 4_8, 4_1_13, 49256, and 7_1 when expressed in either mid-exponential (OD600 = 0.7) or stationary phase (OD600 = 1.2) (Fig.  325   6A). Interestingly, we could not detect any secreted FplA in the spent culture media, as 326 was previously seen for PlpD from P. aeruginosa (Fig. 6B). We then tested for the 327 presence of full length FplA in 10953 (cleaved) and 23726 (uncleaved) in early 328 exponential growth (OD600 = 0.2) and found that we could detect full length and truncated 329 FplA from 10953, indicating that upon increases in bacterial cell density, FplA is cleaved 330 from the surface by an unknown protein and mechanism (Fig. 6C). It is possible that 331 FplA cleavage from the surface results in an active PLA1 domain that remains 332 associated with the surface until released by undetermined host factors (pH, molecular 333 cues, etc.) while colonizing specific regions of the human body. 334 While the FplA amino acid sequences from the seven tested strains are highly 335 similar (>95% identity), we identified two regions in F. nucleatum 23726 and F. 336 nucleatum 25586 at the intersection of the end of the N-terminal extension and just 337 before the end of the PLA1 domain, which could correspond to potential protease 338 processing sites (Fig. 6D, Fig. S6). The suspected cleavage site in F. nucleatum 23726 339 and F. nucleatum 25586 flanking the PLA1 domain is switched from a highly-charged 340 motif (consensus sequence: KNIEDKKEKF), to a more neutral motif (consensus 341 sequence: KFVTNSDAKI) that could be more protease resistant, resulting in retention of 342 the full-length protein. In addition, to arrive at the 25 kDa product seen in five strains, a 343 second cleavage event could occur at the end of the N-terminal extension, as strains 344 23726 and 25586 differ in this region by substitution of an alanine for charged and polar 345 residues (Fig. S6). 346

FplA binds phosphoinositide signaling lipids with high affinity and could play a 348 role in host colonization and altered signaling. 349
We first demonstrated that FplA is a potent phospholipase with PLA1 activity (Fig. 7A) 350 using artificial fluorogenic substrates. Next, we tested FplA for binding to lipids found in 351 human cells and found that it preferentially binds to human phosphoinositides, as was 352 previously seen when characterizing the homologous enzyme PlpD from P. aeruginosa 29 353 (Fig. S7). Upon incubation with a more diverse and freshly-prepared library of PIs, FplA 354 was found to preferentially bind to PI(4,5)p2, and with even stronger affinity to PI (3,5)p2, 355 and PI (3,4,5)p3 lipids (Fig. 7b). This is consistent with structurally homologous enzymes 356 binding PIs, and implicates a role for this enzyme in an intracellular environment. (Fap2, >300 kDa, Type Va secreted) adhesins that are critical for interaction with the 365 host to initiate entry into cells; in turn critical for the onset of inflammation 2,17 . Upon entry 366 into host cells, very little has been reported about how F. nucleatum is able to establish 367 an intracellular niche. We set out to probe the role of a potential virulence factor that 368 we predicted to have phospholipase activity, thereby providing a potential mechanism 369 for colonization and subversion of the host mechanisms of bacterial clearance. We 370 characterized the gene FN1704, which we have renamed fplA for Fusobacterium phospholipase autotransporter (FplA). Our in vitro studies were focused on identifying 372 tools and methods to characterize Type Vd secreted autotransporters to determine their 373 role in virulence in a diverse set of Gram-negative bacteria; many such autotransporters 374 have been identified in intracellular pathogens 28 . We created a F. nucleatum 23726 375 ΔfplA strain which will allow us to next probe the role of this enzyme through the first in
Unless otherwise indicated, E. coli strains were grown in LB at 37°C aerobically, and F. 440 nucleatum strains were grown in CBHK (Columbia Broth, hemin (5 µg/mL) and 441 menadione (0.5 µg/mL)) at 37°C in an anaerobic chamber (90% N2, 5% CO2, 5% H2). 442 For taxonomy verification of Fusobacterium, PCR amplification of a 1502bp region of 443 the 16S rRNA gene sequence was carried out using the universal primers U8F and 444 U1510R ( Table S3) (Fig. 1B, Fig. S1B). A composite predicted structure was 482 assembled using the predicted phospholipase, POTRA, and beta barrel domain, which 483 has the phospholipase domain exposed on the surface of the bacteria (Fig. S1), which 484 we confirmed biochemically as a recombinant protein in E. coli and a native protein in F. 485 nucleatum. In addition, the modeled FplA phospholipase domain was aligned with ExoU 486 (PDB: 4AKX) and VipD (PDB: 4AKF) (Fig. S2A-B). Active site residues in FplA were identified as S98 and D243, and these were verified by multiple enzymatic and chemical 488 biology methods presented in Fig. 2 and Fig. 3. In close proximity to the active site is 489 the oxyanion hole comprised of three consecutive glycine residues (G69, G70, G71). 490 Graphical representations and alignments of all predicted structures were created using 491 PyMOL Molecular Graphics System, Version 1.7.3 Schrödinger, LLC. 492 493

Cloning of FplA Constructs for Expression in E. coli 494
All primers were ordered from IDT DNA and all plasmids and bacterial strains either 495 used or created for these studies are described in Table S1 (Bacterial Strains), Table  496 S2 (Plasmids), and coli expression of FplA constructs. All constructs were created by using 50 ng of 502 genomic DNA as a template, followed by PCR amplification with primers for each 503 construct described in Table S3 using Q5 High-Fidelity Polymerase (NEB, USA) and a 504 ProFlex PCR System (Applied Biosystems, USA) under the following conditions: 98˚C 2 505 min, (98˚C 20 s, 50-62˚C 20 s, 68˚C 1-4 min) x 6 cycles for 50, 53, 56, 59, 62˚C (30 506 cycles total), and 72˚C 5 min. PCR products were then spin column purified and 507 digested overnight at 37˚C with restriction enzymes described in Table S3. Histidine tag that remains on the expressed protein after residues 1-21 from OmpA are 526 cleaved in the periplasm. This effectively creates an inducible vector for the expression 527 of periplasmic and outer membrane proteins in E. coli that was customized with GC rich 528 restriction sites (NotI, KpnI, XhoI) to facilitate enhanced cloning of AT rich (74%) 529 genomes such as F. nucleatum. Using the pDJSVT86 expression vector, pDJSVT88 530 (OmpA1-27, 6xHis, FplA20-760) was created and shows efficient export of enzymatically 531 active, full-length FplA to the surface of E. coli (Fig. 4). to be greater than 95% pure for all constructs. 557

Antibody Production and Western Blotting to Detect FplA 559
Purified FplA20-431 was used to create a polyclonal antibody in rabbits (New England 560 Peptide, USA). To purify the antibody, FplA20-431 was coupled to CNBr-Activated 561 Sepharose (Bioworld, USA) and Anti-FplA20-431 antisera adjusted to pH 8.0 with 20 mM 562 Tris-HCl was passed through the column to bind FplA20-431 antibodies, followed by MgCl2) was added, and immediately transferred by syringe into a sterile, anaerobic tube 606 via septum for incubation at 37˚C for 20 hours with no shaking. Post outgrowth, cells 607 were spun down at 14 kG for 3 minutes, media removed, and resuspended in 0.1 mL 608 recovery media, followed by plating on CBHK plates with 5 µg/mL thiamphenicol and 609 incubation in an anaerobic 37˚C incubator for two days for colony growth. ~ 5 610 colonies/µg of DNA were achieved, and the fplA gene knockout was verified by PCR 611 specific to the chromosome and catP gene that was incorporated into the genome by 612 the pDJSVT100 KO plasmid (Primers , Table S3). In addition, western blots were used 613 to confirm a loss of FplA protein expression (Fig. 5-6). 614

615
Enzymatic assay design, data collection, and FplA kinetics 616 Initial tests for FplA enzymatic activity were run using the EnzChek Phospholipase A1 617 and EnzChek Phospholipase A2 assay kits (ThermoFisher, USA) at 1 µM and 10 µM 618 FplA20-431 using the manufacturer's protocol (Fig. S3A-B). These assays showed that 619 FplA has PLA1, but not PLA2 activity, which is consistent with data reported for the 620 homologous enzyme PlpD. We then went on to further characterize its activity by 621 developing a continuous kinetic assay using the PLA1 specific substrate PED-A1 622 (ThermoFisher, USA) and determined the full kinetic parameters of FplA with this 623 substrate as reported in Fig. 2 and Fig. S3. In detail, FplA was used at 1 nM in the to obtain the values reported in Fig. 2 and Fig. S3. 654

Characterization of FplA inhibitors 656
We set out to characterize inhibitors that we could use as effective tools to test the role 657 of FplA both in vitro and potentially in vivo by IC50 assays using a variety of inhibitor 658 classes. Inhibitors shown in Fig. 3 and Fig. S4 were Characterization of FplA turnover rates and substrate binding affinities with multiple 794 substrates. Results show that FplA binds longer acyl chains with higher affinity, and 795 that loss of the N-terminal Extension domain (residues 20-59) reduces turnover rate but 796 does not affect substrate binding affinity. 797